1. Technical Field
The subject matter of the present invention relates to bifunctional cyclohexyl DTPA ligands and methods of using these compounds. Specifically, such ligands are useful for radiolabeling proteins with radioactive metals, and can consequently be utilized with respect to radioimmunoimaging and/or radioimmmunotherapy.
2. Background Information
This invention relates to metal chelates and the use of metal-chelate protein conjugates.
Interest in the art of metal chelates and in methods for forming metal chelate-protein conjugates for diagnostic and therapeutic purposes continues. Representative type chelates and conjugates and. methods for forming conjugates are disclosed, inter alia, in U.S. Pat. Nos. 4,454,106, 4,472,509, 4,339,426, in EPA 0 279 307 and in German Patent 1,155,122. Other proteins including antibodies, monoclonal antibodies and fragments thereof, monoclonal antibodies and fragments thereof which have been structurally altered by recombinant DNA techniques (i.e., chimeric antibodies), polyclonal antibodies, antigens, blood proteins, or proteins bound to blood lymphocytes or other cells can also be employed in the formation of conjugates.
A method for synthesis of bifunctional metal chelates for conjugation to proteins involves reduction of amino acid amides to ethylenediamines to form monosubstituted derivatives which are converted to bifunctional ethylenediaminetetraacetic acid (EDTA) chelates by alkylation with haloacetic acid. (Yeh et al., Anal. Biochem. 100: I52 (1979)). Similarly, a monosubstituted diethylenetriamine is synthesized by reaction of ethylenediamine with an amino acid ester and reduction of the resulting amide carbonyl. (Brechbiel et al. Inoro. Chem. 25: 2772-8(1986)). Alkylation of the diethylenetriamine with haloacetic acid produces a monosubstituted bifunctional diethylenetriaminepentaacetic acid (DTPA) chelate.
Another method of synthesis of a bifunctional DTPA involves reaction of a DTPA or EDTA carboxylate with a chloroformate ester to form a reactive anhydride (Krejcarek et al., Biochem. Biophys Res. Commun. 77:581 (1977)). The dianhydride of DTPA used as a bifunctional chelate is prepared by dehydration of the parent DTPA. (Hnatowich et al., Int. J. Appl. Rad. Isot. 33:327 (1982)}. The practice of using an EDTA chelate monosubstituted at the carbon-1 position to better retard the release of metal from chelate in vitro, than the unsubstituted EDTA chelate, has also been reported. (Meares et al., Anal. Biochem. 142:68 (1984)).
The prior art has formed metal-protein chelate conjugates by mixing monosubstituted bifunctional EDTA or DTPA chelates or DTPA anhydrides with proteins followed by reaction with the metal to be chelated. (Krejcarek et al., Biochem. Biophys. Res. Commun. 77:581, (1987); Brechbiel et al., Inorg. Chem. 25:5783 (1986)). Imaging of tumor target sites in vivo with metal chelate conjugated monoclonal antibodies prepared according to these methods has been reported. (Khaw et al., Science 209:295, (1980) Sheinberg et al., Science 215:151, (1982)). Diagnosis of human cancer in vivo using metal chelate conjugated monoclonal antibody has also been reported. (Rainsbury et al., Lancet 2:694 (1983)). The use of chimeric antibodies and advantages thereof have been discussed by Morrison, S.L., Hospital Practice (Office Edition) 24:64-65, 72-74, 77-80 (1989). The potential efficacy of using a linking group within a chelate conjugated protein has also been discussed (Paik et al., J. Nucl. Med. 30:1693-1701 (1989)).
However, attempts to employ the tumor localizing properties of metal chelate conjugated monoclonal antibodies for therapeutic purposes have not found common usage, in part because metals may be (and often are) released from the metal chelate conjugate in vivo and, particularly in the case of radioactive metal salts, may produce undesirable concentrations of toxic radionucleotides in bone marrow or kidney or the like even if the conjugates are rigorously purged of adventitiously bonded metal. A process for purifying metal chelate protein conjugates of adventitiously bonded metals is disclosed in U.S. Pat. No. 4,472,509. The importance of using very strong metal chelates to firmly link radiometals to monoclonal antibodies and of rigorous purification of the conjugates to effect maximal tumor localization and minimize delivery to non-target tissues is discussed in Brechbiel et al., Inorg. Chem. 25:2772-81 (1986)). Undesirable localization of potentially therapeutic radionuclides released in mice in vivo from metal chelate conjugated polyclonal antibodies have precluded some therapy investigations in humans. (Vaughn et al., EIR-Bericht. Vol. 78, (1986)). Increased in vivo bone uptake of radiometal injected for therapy as a metal chelate conjugated monoclonal antibody has also been reported. (Hnatowich et al., J. Nucl. Med. 26:503 (1985)). The amount of potentially therapeutic doses in humans of radiometal chelated polyclonal antibody has been limited by bone marrow toxicity (Order et al., Int. J. Rad. Oncol. 2:277 (1986)). Kidney uptake of radiometal has recently been reported as preventing human use. (Macklis et al, Science. 240:1024-26 (1988)].
Disubstituted bifunctional DTPA derivatives have proven useful for labeling of proteins with radioactive metals (Kozak, et al., Cancer Research 49:2639-44 (1989)). The introduction of a second substituent on the carbon backbone of DTPA was seen to retard the loss of metal from the DTPA ligand when linked to antibody and injected into the circulation of animals.
The usefulness of radionuclide materials in cancer therapy is disclosed in the article, Kozak et al., "Radionuclide-conjugated monoclonal antibodies: A Synthesis of Immunology, in Organic Chemistry and Nuclear Science" Trends in Biotechnoloqy. 4(10):259-64 (1985). This article discusses the use of antibody conjugates to deliver either alpha or beta radiation. The value of alpha radiation for bismuth-212 in radionuclide therapy is further discussed in the two articles, Kozak et al., "Bismuth-212-labeled anti-Tac monoclonal antibody: Alpha-particle-emitting Radionuclides as Modalities for Radiommunotherapy," Proc. Natl. Acd. Sci. U.S.A. 83:474-478 (1986) and Gansow et al., "Generator-produced Bi-212 Chelated to Chemically Modified Monoclonal Antibody for Use in Radiotherapy," Am. Chem. So. Symposium Series 15:215-227 (1984). Ligands, for the secure linkage of bismuth to proteins, have not been available. (Macklis et al., Science 240:1024-2 (1988)).
Examples of other uses for chelated metal ions are disclosed in the following articles. Magerstadt et al., "Gd(DOTA): An alternative to Gd(DPTA) as a T.sub.178 Relaxation Agent for NMR Imaging or Spectroscopy," Magnetic Resonance in Medicine 3:808-812 (1986), discloses the usefulness of gadolinium as a relaxation agent for NMR imaging. The article, Spirlet et al., "Structural Characterization of a Terbium (III) Complex with 1,4,8,11-Tetraazacyclotetradecane- 1,4,8,11 tetraacetic acid. Lanthanide Ions and Confirmation of the 14- Membered Ring," Inorgan. Chem. 23:4278-4783 (1984), disclosed the usefulness of lanthanide chelates.
All patents and publications referred to herein are hereby incorporated by reference.
It is evident from the above that there continues to be a need for more effective metal chelate protein conjugates that firmly link metals to proteins to minimize metal release and permit highly selective delivery of metals to targeted sites in vivo.